All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
Predatory marine snails of the genus Conus (cone snails) are a highly diverse family of marine molluscs which capture their prey by envenomation (Screenivasan, 2002). The venom of a typical cone snail is a complex mixture comprising about one hundred different peptides, which target different ion channels and receptors and interfere with their function, resulting in immobilisation of the prey (Olivera et al, 1990; Olivera and Cruz, 2001).
The mixture of peptides present depends on the species of cone snail and the prey on which it feeds, and may vary with time even in individual molluscs (Lewis et al 1994; Jones et al 1996; Bingham et al 1996). Classes of peptides found in Conus venoms include the α-, αA- and ψ-conotoxins (antagonists at nicotinic acetylcholine receptors), μ-, μO-conotoxins (antagonists at voltage-gated sodium ion channels), δ-conotoxins (agonists, which inhibit inactivation at voltage-sensitive sodium channels), ω-conotoxins (antagonists at voltage-sensitive calcium channels), κ- and κA-conotoxins (antagonists at potassium channels), σ-conotoxins (inhibitors of 5HT receptors), χ-conotoxins (inhibitors of noradrenaline uptake), conantokins and conodynes (antagonists at NMDA receptors), ρ-conotoxins (inhibitors of the α1-adrenoceptors), conorfamides (Maillo et al., 2001), and contulakins (inhibitors of neurotensin receptors). For review see Jones and Bulaj, 2000; McIntosh and Jones, 2001).
The α-conotoxins are typically found in cone snails which prey on fish or on marine snails or marine worms. They are typically 12-19 amino acids in length, and have four cysteine residues which form two disulfide bonds, forming a two-loop structure (McIntosh et al, 1999). They are characterised by an ability to inhibit the nicotinic acetylcholine receptor (nAChR). nAChRs are ligand-gated ion channels which consist of five subunits arranged around a cation-conducting pore (Sargent 1993; Lukas et al 1999; Karlin, 2002).
There are two main classes of nAChRs:
1) the neuronal type; and
2) the muscle type.
Neuronal type nAChRs are present both pre- and post-synaptically in the central and peripheral nervous systems, while the muscle type nAChRs are found post-synaptically at skeletal neuromuscular junctions (Wonnacott 1997). The main difference between these receptors is their subunit composition. The neuronal type receptors are formed from the combination of α and β subunits or α subunits alone, while the muscle type receptors are composed of α, β, γ and ε (or δ) subunits. The functional receptors have different combinations of subunits (see Karlin, 2002), and have a range of pharmacological properties (see Albuquerque et al., 1997; Lukas et al 1999 for review). Specific α-conotoxins display different affinity and selectivity for muscle and neuronal nAChRs and their subtypes.
Compounds of the α-conotoxin class may be useful in the treatment of disorders which involve the neuronal nAChR. The neuronal nAChR has been implicated in the pathophysiology of Alzheimer's disease (Guan et al, 2000), Parkinson's disease (Aubert et al, 1992), schizophrenia (Mukherjee et al, 1994), small cell lung carcinoma (Codignola et al, 1996) nicotine addiction (U.S. Pat. No. 5,780,433, U.S. Pat. No. 5,866,682), pain (Marubio et al 1999), and as neuromuscular blocking agents, such as muscle relaxants (U.S. Pat. No. 6,268,473 and U.S. Pat. No. 6,277,825) and in certain forms of epilepsy (Steinlein et al 1995).
Conotoxins of another class, the ω-conotoxin class, have provided lead compounds for stroke and for pain. ω-conotoxin MVIIA (Neurex SNX-111, Warner-Lambert CI-1009, or Elan's Ziconotide) and ω-conotoxin CVID (AMRAD AM336) are presently undergoing clinical trials for the treatment of manifestations of stroke (Zhao et al 1994; Heading, 1999; Shen et al 2000; Jones and Bulaj, 2000) and for chronic pain (Bowersox et al 1996; 1997; Jain, 2000; Jones and Bulaj, 2000). These compounds target N-type calcium channels in nerves. However, the members of the ω-conotoxin class still have undesirable side effects in some patients (Penn and Paice, 2000), and the US Food and Drug Administration has requested a repeat of the Stage III clinical trials for Ziconotide (re-named Prialt™ by Elan) for treatment of cancer pain.
Yet another class of conopeptide, the conantokins, having 10-30 amino acids, including preferably two or more γ-carboxyglutamic acid residues, have been developed for the treatment of neurological and psychiatric disorders, including pain, e.g., as an analgesic agent (U.S. Pat. No. 6,277,825). In addition, a novel class of conopeptide, whose target receptor is yet to be defined (McIntosh et al 2000), has been shown to have analgesic activity in the mouse.
We have recently found that a specific amino acid at position 10 (Leu10) of α-conotoxin PnIB is responsible for conferring potency for the neuronal-type nicotinic response (Broxton et al 2000). We have also found that splice variants of the α-conotoxins PnIA and PnIB from Conus pennaceus show improved selectivity for α7 subunits of nAChRs (U.S. patent application Ser. No. 09/639,565; see also Hogg et al, 1999; Broxton et al, 2000).
We have now found that a previously unexamined Conus species, Conus victoriae, which is found along the north-western coast of Australia, has novel α-conotoxins with unexpectedly powerful analgesic activity. Surprisingly, the ability of one particular α-conotoxin (Vc1.1) to inhibit sensory nerve function, and consequently its analgesic activity, is even higher than that of ω-conotoxin MVIIA (ziconotide, Prialt™) from Conus magus. In addition, we have found that a post-translational modification of this peptide lacks analgesic activity but retains the ability of the parent compound to accelerate recovery from nerve injury. We have also identified two other new α-conotoxins, conotoxin An1.1 from Conus anemone and conotoxin Vg1.1 from Conus virgo, which have similar sequences to Vc1.1, and have similar pharmacological actions.